The invention relates to analytical methods and devices for the detection of feline leukemia virus (FeLV) and antigens thereof in the saliva of cats. More particularly, the invention relates to methods and devices for acquiring saliva samples and to immunoassay procedures and kits for detecting the presence of FeLV and FeLV antigens in saliva samples.
The spread of feline leukemia caused by FeLV poses a significant health problem in many cat populations. There is evidence that horizontal transmission of FeLV within cat populations contributes significantly to the occurance of feline leukemia. Transmission of FeLV may cause transient or persistant viremia, which may then lead to leukemia and/or various other fatal pathologies. Evidence suggests that the saliva of viremic cats can carry infectious virus and that cat saliva may serve as an agent for the horizontal transmission of FeLV.
The detection of FeLV viremia is useful for identifying cats which may develop leukemia or other pathologies associated with FeLV. Cats having a FeLV viremia may have a diminished economic value. Additionally, some cat owners may wish to isolate infected cats for observation and to eliminate cats which are persistant excretors of FeLV and FeLV antigens. Eliminations of the persistant excretors diminishes the risk or horizontal transmission of FeLV into population clusters.
Several serum assays have been developed for the detection of FeLV and FeLV antigens in serum. The LEUKASSAY (TM) is a micro ELISA test based on the principle of the double antibody sandwich technique. The LEUKASSAY (TM) is marketed by Pitman-Moore, Inc., Washington Crossing, New Jersey, U.S.A. and is described by A. S. Mia, D. E. Kahn, M. M. Tierney and J. E. Post (Comparative Immunology, Microbiology and Infectious Diseases, vol. 4(1), pp. 111-117 (1981). The test procedure consists of a first incubation of 50 microliters of serum sample in the polyclonal anti-FeLV antibody-coated well of a micro ELISA plate; a second incubation of polyclonal anti-FeLV antibody peroxidase conjugate within the well; and finally, washing unbound conjugate from the well and incubating with chromogenic substrate so as to indicate the presence of bound conjugate within the well. A substantially identical serum assay is described by Carl Saxinger (Intervirology, vol. 15, pp. 1-9 (1981)).
An improved serum assay is described by Hans Lutz, Neils C. Pedersen, and Gordon H. Theilen (American Journal of Veterinary Research, vol. 44(11), pp. 2054-2059 (1983)). The improved serum assay may be used, in conjunction with other tests, for the detection of immune carriers of FeLV. The improved serum assay employs monoclonal antibody having a specificity for FeLV antigen p27, a nucleocapsid protein antigen of FeLV. The p27 monoclonal antibody is incorporated into a micro ELISA for the analysis of FeLV and FeLV antigens in serum. When used in conjunction with other tests, the monoclonal micro ELISA is helpful in the identification of immune carriers of FeLV.
A number of studies have investigated the presence and properties of FeLV in saliva. The stability and infectivity of FeLV in cat saliva dried onto glass surfaces was described by Donald P. Francis, M. Essex, and Dawn Gayzagian (Journal of Clinical Microbiology, vol. 9(1), pp. 154-156 (1979)). In this study, drooled saliva was collected into an iced petri dish from a FeLV excretor cat narcotized with ketamine hydrochloride and induced to salivate by the application of a drop of atropine onto the tongue. The infectivity of FeLV was compared for freshly drooled saliva and for saliva smeared and dried on a glass surface.
Other studies have employed more direct techniques for the collection of saliva excretions. A collection method employing Dacron (TM) swabs was described by Edward A. Hoover, Richard G. Olsen, Lawrence E. Mathes, and Joseph P. Schaller (Cancer Research, vol. 37, pp. 3707-3719 (1977)). This collection method employed Dacron (TM) swabs to obtain material from the oropharynx. After swapping the oropharynx, the collected material is then expressed from the Dacron (TM) swabs using 1 ml. of Hanks balanced salt solution.
A substantially identical technique was described by D. P. Francis et al. (Journal of Clinical Pathology, vol. 32(5), pp. 4514-4515 (1979); Leukemia Research, vol. 3(6), pp. 435-441 (1979); and Nature, vol. 269, pp. 252-254 (1977)). Francis et al. describe the use of calcium alginate swabs (purchased from Calgiswabs, Inolex, Glenwood, Illinois, U.S.A., but no longer available at that source). Francis et al. demonstrate that there is a quantitative correlation between the FeLV and FeLV antigens found in saliva collected by means of rotating a calcium alginate swab within the cat's buccal crease and the FeLV and FeLV antigens found in saliva collected by drooling into an iced petri dish and pipetted therefrom. After rotation in the buccal crease, the calcium alginate swab should be frozen until the FeLV and FeLV antigens are measured. The calcium alginate swabs are cut and placed in sterile vials containing 1.8 ml McCoy's 5A medium with 15% fetal bovine serum, 100 IU/ml penicillin, 100 micrograms/ml streptomycin, and 0.25 micrograms/ml amphotericin. The vials are then frozen at -90.degree. C., until tested. When thawed, the vials are mechanically agitated and the supernatant is diluted and overlaid onto indicator cells. The calcium alginate swabs have been shown to absorb a constant volume of saliva and allow quantification of the presence of FeLV and FeLV antigens.
Saliva analytes, including FeLV and FeLV antigens, can also be assayed using immunochemically reactive dip sticks. U.S. Pat. No. 4,444,880 (Henry Tom, Apr. 24, 1984) discloses a dip stick having a bibulous support for analyzing saliva samples. The Tom device includes a bibulous support which is impregnated with a composition that prevents interference with the immunoassay. Further dip sticks are disclosed in U.S. Pat. No. 4,135,884 (James Shen, Jan. 23, 1979) and in U.S. Pat. No. 4,305,924 (Piasio et al., Dec. 15, 1981). Both the Shen and Piasio devices could also be employed for assaying saliva analytes, including FeLV and FeLV antigens. However, in both cases, the saliva sample must first be collected and then added to the assay vessel. Neither the Shen nor the Piasio dip stick device can be employed to collect the saliva sample directly from the donor oral cavity.
What is needed is a simple method which employs an immunochemically reactive dip stick to collect a saliva sample directly from the donor's oral cavity and then to assay or test for the presence of FeLV, FeLV antigens, and other saliva borne antigens and analytes directly on the same dip stick by the development of a color reaction.